Generating kidney organoids and mature renal cell types via directed iPSC differentiation. Chronic kidney disease is increasing in prevalence by 6.3% per annum. Hence there is a desperate need for the development of novel treatments. The aim of the proposal is to direct the differentiation of human pluripotent stem cells towards kidney organoids containing all four key progenitor cells capable of generating a mature organ, including the nephrogenic mesenchyme, ureteric epithelium, stroma and vascular progenitors. These organoids will serve as a source of specific kidney cell types of value for cellular therapy as well as an accurate model of the organ as a whole. The proposal draws on our published data defining the methodology for generating kidney organoids and our experience in the generation of reporter iPSC lines. Results from the studies proposed will generate tools for the research community (a suite a reporter cell lines), knowledge about the development of the kidney in humans, improved methods for differentiation and proof of concept data for the application of the resulting cells in drug/nephrotoxicity screening and cellular therapy. The validity of the kidney organoids as a model of the developing organ will be interrogated using expression profiling, including single cell RNA-seq analyses. Single, double and triple fluorescent reporter iPSC lines will be generated to facilitate the subsequent isolation of specifi cell types from the organoids post differentiation. These cell types will also be characterized, providing novel information about specific kidney cell types during human development. Validating the use of organoids as a model of the developing organ will be performed so as to develop the use of organoids for drug/nephrotoxicity screening. Reporter iPSC lines will also be generated to report defined responses to injury. Nephrotoxicity screening will be performed on organoids generated using such reporter. Specific kidney cell types will be isolated from organoids via FACS and used to evaluate cellular therapy approaches for the treatment of chronic kidney injury via transplantation into immunocompromised mice. Reporter iPSC lines will also be generated to incorporate the expression of luciferase to allow for the monitoring via bioluminescence of the location of the delivered cells in live animals across time. The efficacy of delivering kidney cells derived from pluripotent stem cells into preclinical models will therefore be determined.